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  • Based on the improved characteristics of the mutant ECDs


    Based on the improved characteristics of the mutant ECDs we proceeded to crystallization trials, starting with the β1-BPloop as the most promising. We obtained rod shaped hexagonal crystals reproducibly, but with moderate diffraction ability (Fig. 5). Furthermore, the crystals were sensitive in mounting loop manipulations and temperature changes indicating a high water content, which could be responsible for the low diffraction ability. Extensive optimization is underway with respect to crystal growth, cryoprotective conditions and manipulations, while crystal dehydration and humidity control upon data collection are also factors that need to be considered, aiming to obtain crystals suitable for high resolution diffraction data. Overall, the proteins designed and presented herein pose as tools for both the investigation of MG antibody binding and structural studies. The profound improvement of the solubility of the AChR ECDs expressed in yeast has led to the production of crystals of the β1 ECD, although further improvement is required. Furthermore, the great increase in expression yields achieved compared to the wild type proteins, allows their use for large-scale applications, such as antibody adsorbents in a novel therapy for MG [34].
    Acknowledgments This work was supported by grants from the European Commission (FP7 Fight-MG, Contract No. 242210 and FP7 REGPOT-2010-1 NeuroSign, Contract No. 264083) and the Greek NSRF (“Thalis”).
    Introduction The autonomic nervous system regulates important visceral and homeostatic function such as blood pressure, pupil responses, gastrointestinal motility and bladder dysfunction. Ganglionic SMIP004 receptors (AChR) mediate fast transmission within autonomic ganglia, and transgenic mice that lack ganglionic AChR have severe autonomic failure. An important cause of acquired dysautonomia in adults is autoimmune autonomic ganglionopathy (AAG), which is due to auto-antibodies that bind to the alpha 3 subunits of ganglionic AChR. Ganglionic AChR antibodies do not typically cross react with muscle acetylcholine receptors, the common antigen in autoimmune myasthenia gravis, and are not found in healthy controls. AAG typically presents subacutely in middle or old age with pandysautonomia. We report an adolescent with a subacute pandysautonomia associated with antibodies against ganglionic AChR, who improved after treatment with immune suppressive therapies.
    Method Antibodies to α3 ganglionic form of the acetylcholine receptor (AChR) are measured by a radioimmunoprecipitation of 125I-epibatidine-labelled α3-AChR. In short, IMR32 human neuroblastoma cell line is grown in culture (DMEM, 10% FCS) until confluent and then harvested. The cells are washed twice in phosphate buffered saline and extracted in 0.05 M Tris–HCl pH 7.2 containing 2% Triton X100. The cell pellet is removed by centrifugation (13,000 rpm, 15 min, 4 °C) and the supernatant labelled 125I-epibatidine (Perkin Elmer, USA) for 2 h at room temperature. Test and control sera are incubated overnight at 4 °C with the labelled extract and then the complex is immunoprecipitated by the addition of sheep anti-human IgG (Binding Site, Birmingham, UK). Immunoprecipitation of this 125I-epibatidine-labelled α3-AChR extract can be achieved by antibodies to α3-AChR but not by antibodies to α4-AChR or α7-AChR (sigma Chemical Company, UK).
    Case report
    Discussion Subacute pandysautonomia is rare in children and α3-AChR antibodies can differentiate AAG from other causes of pandysautonomia such as diabetic autonomic neuropathy and idiopathic gastrointestinal dysmotility syndromes. It is probable that children with AAG have been previously described, but this is the first report to confirm the presence of α3-AChR antibodies in childhood AAG. Adults with AAG associated with α3-AChR autoantibodies typically present subacutely with pandysautonomia resulting in impairment of sympathetic function (orthostatic hypotension, anhidrosis), parasympathetic function (dry mouth, dry eyes, abnormal pupil constriction), enteric function (gut dysmotility) and bladder dysfunction. Adults with AAG are more likely to be seropositive for ganglionic α3-AChR antibodies if they have a subacute pandysautonomica with abnormal pupillary responses, sicca complex and gut dysautonomia. Our patient with positive ganglionic α3-AChR antibodies had a syndrome of pandysautonomia referable to multiple organs. By contrast two other adolescent patients with isolated gut pseudo-obstruction as part of systemic lupus erythematosus (SLE) have been tested and are seronegative for ganglionic α3-AChR antibodies (titre 16 and 19 pM, normal < 100 pM). However, patients with seronegative autonomic ganglionopathy may also gain benefit from immune therapies, and it is proposed that these patients may harbour other unidentified autoantibodies or have alternate autoimmune processes.